The terminal two enzymes of the heme biosynthetic pathway, protoporphyrinogen oxidase and ferrochelatase, catalyze the conversion of protoporphyrinogen to protoporphyrin followed by insertion of ferrous iron to form the end product protoheme. These enzymes are of general biochemical interest for not only their unique catalytic functions, but also because of their vectoral organization across the membrane. Medically they are of interest since decreased activities of either one results in a disease condition known generally as porphyria. In addition it has been shown recently that drug induced heme destruction in cytochrome P450 can lead to a porphyric condition in healthy animals and that the mechanism of this porphyria involves in vivo inhibition ferrochelatase. The proposed research is designed to increase our understanding of ferrochelatase and protoporphyrinogen oxidase. Specifically the data gathered will include information about protein sequence, membrane topology of the proteins, details of protein-protein interactions and the mechanism of substrate and product transport into and out the mitochondrion. An additional benefit of the experimental approaches to be exploited in this study will be the design of ELISA assays to quantitate both ferrochelatase and protoporphyrinogen oxidase. These procedures should prove to be of general medical use in future screening for varigate porphyria and protoporphyria. The major experimental approaches can be broken down into four categories. These are: 1) examination of possible protein-protein interactions between ferrochelatase and protoporphyrinogen oxidase, 2) determination of the membrane topology of these two enzymes, 3) amino acid sequence determination of putative active site peptides for both enzymes and eventual total amino acid sequence for ferrochelatase and 4) examination of porphyrin and heme movement across the inner mitochondrial membrane.